The present invention relates to a vaccine composition for the immunocontraception of fish. The present invention also relates to a vaccine composition for the immunocontraception of birds.
Among vertebrates, mating strategies involve behaviour, gamete structure and the specificity of recognition of sperm and egg. Mammalian oocytes are surrounded by an envelope called the zona pellucida that is composed of three glycoproteins in a ratio of 1:2:2 denoted by ZPA, ZPB, and ZPC (Harris, J. D., Hibler, D. W., Fontenot, G. K., Hsu, K. T., Yurewicz, E. C. and Sacco, A. G.(1994) xe2x80x9cCloning and characterization of zona pellucida genes and cDNA""s from a variety of mammalian species: the ZPA, ZPB and ZPC gene families DNA sequence.xe2x80x9d J. Sequencing and Mapping 4:361-393). The zona pellucida contains species-specific sperm receptors composed mainly of O-terminal oligosaccharides. Fish eggs have a teleost equivalent of mammalian zona pellucida wherein the carbohydrate moiety has some structural similarity to the carbohydrate moiety of mammalian zona pellucida (Taguchi, T., Seko, A., Kitajima, K., Muko, Y., Inoue, S., Knoo, K-H., Morris, H. R., Dell, A. and Inoue, Y.(1994) xe2x80x9cStructural studies of a novel type of pentaantennary large glycan unit in the fertilization-associated carbohydrate-rich glycopeptide isolated from the fertilized eggs of Oryzias latipes.xe2x80x9d J. Biol. Chem 269:8762-8771).
Mouse ZP2 (ZPA) contains a 241-amino acid domain at the C-terminus with 28% identity with a white flounder teleost egg protein (Lyons, C. E., Payette, K. L., Price, J. L. and Huang, R. C. C. (1993) xe2x80x9cExpression and structural analysis of a teleost homolog of a mammalian zona pellucida gene.xe2x80x9d J. Biol. Chem. 268:21351-21358). A 348-amino acid sequence of mouse ZP1 (ZPB) is 47% similar (32% identical) to that of mouse ZP2 (ZPA) suggesting that this protein domain has been duplicated in mammals (Epifano, O., Liang, L-F. and Dean, J. (1996) xe2x80x9cMouse ZP1 encodes a zona pellucida protein homologous to egg envelope proteins in mammals and fish.xe2x80x9d J. Biol. Chem. 270: 27254-27258). A smaller region of this sequence (275 amino acids) is 52% similar (36% identical) with a white flounder egg envelope protein that contains 509 amino acids.
Immunization of grey seals with a single administration vaccine containing soluble zona pellucida antigens encapsulated in liposomes has been shown to reduce female fertility by at least 90% for up to at least six years (Brown, R. G., Kimmins, W. C., Mezei, M., Parsons, J. L., Pohajdak, B. and Bowen, W. D. (1996) xe2x80x9cBirth control for grey seals.xe2x80x9d Nature 379:30-31; Brown, R. G., Bowen, W. D., Eddington, J. D., Kimmins, W. C., Mezei, M., Parsons, J. L., and Pohajdak, B. (1997) xe2x80x9cEvidence for a long-lasting single administration contraceptive vaccine in wild grey seals.xe2x80x9d J. Reproduct. Immunol. 35:43-51; and U.S. Pat. No. 5,736,141). The same vaccine prevented pregnancy in four rabbits (proven breeders) following 8 matings (unpublished observations).
An example of the use of liposome encapsulation of denatured recombinantly produced protein to raise antibodies against a native protein was shown with Neisseria meningitidis outer membrane protein P1. (Muttilainen, S., Idanpaan-Heikkila, I., Wahlstrom, E., Nurminen, M., Makela, P. H. and Sarvas, M. (1995) xe2x80x9cThe Neisseria meningitidis outer membrane protein P1 produced in Bacillus subtilis and reconstituted into phospholipid vesicles elicits antibodies to native P1 epitopes.xe2x80x9d Microb. Pathog. 18:423-436).
Specificity of recognition of sperm and egg is essential in any species. However, the mechanism of fertilization varies widely, both physiologically and biochemically, between species. Fertilization in fish differs from that in mammals in that most teleostean fish spermatozoa lack an acrosomal structure. Penetration by a spermatozoon of the fish egg envelope occurs via a discrete micropyle with closure of the micropyle after penetration of the first spermatozoon.
Sperm-egg interaction in birds is significantly different from that in mammals and different again from fish. In birds, sperm-egg recognition is initiated by the binding of spermatozoa to the inner perivitelline layer (IPVL), a proteinaceous structure surrounding the avian ovum (Bakst, M. R. and Howarth, B. (1977) xe2x80x9cHydrolysis of hens perivitelline layer by cock sperm in vitro.xe2x80x9d Biol. Reproduct. 17:370-379). There is no block to polyspermy in avian species but a further proteinaceous layer, the outer perivitelline layer (OPVL), is laid down about 15 minutes after the IPVL in chickens and appears to prevent further penetration of sperm. Therefore, if spermatozoa can be prevented from entering the avian egg between the laying down of the IPVL and OPVL, by antibodies directed against the IPVL, then immunocontraception would be realized.
There is some similarity between reproduction in mammals and fish but also many differences. Unlike the C-terminus, the N-terminus domain of white flounder egg protein is quite dissimilar to mouse ZP2 (ZPA) and a transmembrane domain characteristic of all mammalian zona pellucida proteins is not present in teleost egg protein indicating the divergence of these species 650 million years ago (Epifano, O., Liang, L-F. and Dean, J. (1996) xe2x80x9cMouse ZP1 encodes a zona pellucida protein homologous to egg envelope proteins in mammals and fish.xe2x80x9d J. Biol. Chem. 270: 27254-27258).
The carbohydrate moiety of teleost egg glycoproteins is also dissimilar, for example, rainbow trout egg envelope glycoprotein has a unique N-linked glycan containing KDN (2-keto-3-deoxy-D-glycero-D-qalacto-nononic acid) in the second layer of the vitelline envelope (Tezuka, T., Taguchi, T., Kanamori, A., Muto, Y., Kitajima, K., Inoue, Y. and Inoue, S. (1994) xe2x80x9cIdentification and structural determination of the KDN-containing N-linked glycan chains consisting of bi- and triantennary complex-type units of KDN-glycoprotein previously isolated from rainbow trout vitelline envelopes.xe2x80x9d Biochem. 33:6495-6502). This KDN-glycoprotein is exposed to the outer surface around the micropyle through which sperm enter the egg at fertilization. Most fish sperm lack an acrosome and penetrate the fish egg envelope via a discrete micropyle. The micropyle forms a guidance system in teleost fertilization that enhances sperm penetration (Amanze, D. and Iyengar, A. (1990) xe2x80x9cThe micropyle: a sperm guidance system in teleost fertilization.xe2x80x9d Development 109:495-500). A chemical attractant may also emanate from the micropyle to enhance the chance of fertilization.
In spite of the significant structural differences between fish egg envelope protein and mammalian zona pellucida, fish egg envelope proteins have been designated the teleost homolog of zona pellucida (TH-ZP for convenience of reference). In fish, TH-ZP3 is made in the liver and transported via the blood to the ovary, while TH-ZP2 is made in the ovary (Hamazaki, T. S., Nagahama, Y. and Yamagami, K. (1989) xe2x80x9cA glycoprotein from liver constitutes the inner layer of the egg envelope (zona pellucida interna) of the fish, Oryzias latipes.xe2x80x9d Dev.Biol.133:101-110; Murata, K., Sasaki, T., Yasumasu, S., Iuchi, I., Enami, J., Yasumasu, I. and Yamagami, K. (1995) xe2x80x9cCloning of cDNAs for the precursor protein of a low-molecular weight subunit of the inner layer of the egg envelope (chorion) of the fish Oryzias latipes.xe2x80x9d; Chang, Y. S., Wang, S. C., Tsao, C. C. and Huang, F. L. (1996) xe2x80x9cMolecular cloning, structural analysis and expression of carp ZP3 gene.xe2x80x9d Mol. Reprod. Dev. 44:295-304; Murata, K., Sugiyama, H., Yasumasu, S., Iuchi, I., Yasumasu, I. and Yamagami, K. (1997) xe2x80x9cCloning of cDNA and estrogen-induced hepatic gene expression for chorigenin H, a precursor protein of the fish egg envelope (chorion).xe2x80x9d Proc.Natl.Acad.Sci. USA 94:2050-2055; Chang, Y. S., Hsu, C. C., Wang, S. C., Tsao, C. C. and Huang, F. L. (1997) xe2x80x9cMolecular cloning, structural analysis and expression of carp ZP2 gene.xe2x80x9d Mol. Reprod. Dev. 46:258-67).
It is undesirable that transgenic fish escape from fish farms and mate with fish in the wild. This problem would be reduced if females were sterile. Such sterile fish could also redirect their food reserves to increase their body size rather than roe production. Triploid fish are sterile but triploid salmon grow poorly (MacKenzie, D. (1996) xe2x80x9cCan we make supersalmon safe?xe2x80x9d New Scientist pp 14-15). Triploidy can be induced in fish by a pulse of pressure that prevents embryos from ejecting one set of chromosomes.
With respect to birds, population control of certain species is of great environmental importance. For example, some Canada geese (Branta canadensis) populations in the USA, Canada and Europe have increased to a point that threatens other bird populations and are a nuisance to the enjoyment of parks, golf courses, etc. Burgeoning populations of snow geese (Chen caerulescens) are wreaking havoc on precious tundra habitat (Struzik, E. (1998) xe2x80x9cThe snow geese dilemma.xe2x80x9d Equinox 97:50-57) and have resulted in compensation claims in Quebec, Canada alone of $844,000 in 1996. Some tundra habitats have been described as 35% overgrazed, 35% damaged and 30% destroyed by snow geese. In addition, many populations of small birds such as pigeons (Columba livia) and starlings (Sturnus vulgaris) cause significant economic loss in many parts of the world. As a consequence, there is need for management of some bird populations.
The present invention provides a single administration immunocontraceptive for fish.
More specifically, the present invention provides an immunocontraceptive vaccine composition comprising a teleost homolog of zona pellucida (TH-ZP), together with a pharmaceutically acceptable diluent or carrier, for preventing fertilization in a fish.
In another aspect, the present invention provides a method for preventing fertilization in a fish comprising administering an effective amount of the composition of the invention, comprising a teleost homolog of zona pellucida (TH-ZP), to the fish.
It is preferred that an adjuvant, such as Freund""s complete adjuvant (FCA) or another biologically acceptable adjuvant, be present to assist in stimulation of an immune response in fish. It is also preferred that the TH-ZP be encapsulated into a liposome for administration. Preferably the liposome is multilamellar and comprises L-xcex1-lecithin (soybean) and cholesterol, since this will effect slow release of TH-ZP resulting in an extended period of antibody production and thereby an extended period of contraception in fish. In addition, antibodies raised by this immunological procedure will be directed to the native protein antigens.
The present invention also provides a single administration immunocontraceptive for birds.
Accordingly, in another aspect, the present invention provides an immunocontraceptive vaccine composition comprising an antigen from an inner perivitelline layer (IPVL) of a bird egg, together with a pharmaceutically acceptable diluent or carrier, for reducing or preventing fertilization in a bird.
In another aspect, the present invention provides a method for preventing fertilization in a bird comprising administering an effective amount of the composition of the invention, comprising the antigen from an inner perivitelline layer (IPVL), to the bird.
It is preferred that an adjuvant, such as Freund""s complete adjuvant (FCA) or another biologically acceptable adjuvant, be present to assist in stimulation of an immune response in birds.
It is preferred that the antigen from the IPVL, e.g. in an IPVL portion, be encapsulated into a liposome for administration. Preferably the liposome is multilamellar and comprises L-xcex1-lecithin (soybean) and cholesterol, to effect slow release of antigen/IPVL and increase production of antibodies that bind to the target proteins. This will result in an extended period of antibody production and thereby an extended period of contraception in birds.
As well as FCA, other adjuvants that can be used in vaccine compositions of the present invention include non-ulcerative Freund""s complete adjuvant, Freund""s incomplete adjuvant, TITERMAX(trademark), MF89, Gerbu, Bacillus Calmette-Guerin, RIBI (MPL+TDM+CWS), bacterial lipopolysaccharide, sodium phthalate derivative of bacterial lipopolysaccharide, sodium phthalate derivative of lipopolysaccharide plus alum, SUPERCARRIER(trademark), ADJUPRIME(trademark) and Alum.
In general, any suitable liposome can be used in the fish or bird vaccine compositions disclosed herein. Anionic and neutral liposomes are well-known in the art (see, e.g., Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), for a detailed description of methods for making liposomes) and are useful for delivering a large range of products.
Cationic lipids are also known in the art. Such lipids include Lipofectin(trademark) also known as DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2-bis(oleyloxy)-3-(trimethylammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Chol (3 beta-(N-(Nxe2x80x2, Nxe2x80x2-dimethyl aminomethane)-carbamoyl) cholesterol). A description of these cationic lipids can be found in EP 187, 702, WO 90/11092, U.S. Pat. No. 5,283,185, WO 91/15501, WO 95/26356, and U.S. Pat. No. 5, 527, 928.
The route of administration of the vaccine compositions disclosed herein can be any route used typically used in the vaccine field. As general guidance, administration can be via a mucosal surface, e.g., an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface; or via a parenteral route, e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route. The choice of administration route depends on the formulation that is selected as well as on the animal to be vaccinated.
Administration is achieved in a single dose or repeated as necessary at intervals, as can be determined readily by one skilled in the art. An appropriate dose depends on various parameters including the recipient (e.g., adult or infant), the particular vaccine antigen, the route and frequency of administration and the presence/absence or type of adjuvant as can be determined by one skilled in the art.
It should be noted that all of the antibody titers referred to in the specification are measured in comparison with the antibody titer in a reference serum. The titer in the reference serum was arbitrarily assigned a value of 100. That value has no relationship to the absolute titer required to produce an immunocontraceptive effect. In fact, titers of only a few percent of those found in the reference serum are sufficient to produce an immunocontraceptive effect in some cases. While the reference serum clearly contains sufficient antibody to effect immunocontraception, it does not represent an indication of the minimum antibody titer needed for immunocontraception.